The tegument region of herpesviruses is located between the outer surface of the capsid and the inner surface of the envelope and is made up of approximately 20 different proteins. Recent evidence suggests that the tegument is not a static structure and certain dynamic interactions may occur among tegument proteins within the assembled virion. The long-term objective of our research program has been to define molecular events associated with the assembly of the tegument of herpes simplex virus type 1 (HSV-1). The specific objective of this application is to define the dynamic events associated with the assembly of the HSV-1 abundant tegument protein designated UL46 (VP11/12). We have recent evidence that UL46 undergoes a cleavage event that results in both full-length UL46 and an amino-terminal truncation of UL46, designated t-UL46, being associated with assembled virions. We have also shown that amino-terminal truncated mutants of UL46 traffic to the nucleus, possibly due to the unmasking of a conserved nuclear localization signal at the carboxyl end of the protein. Our working hypothesis is that cleavage of virion-associated UL46 during the assembly of the virion, enables the t-UL46 that is released from the incoming virus to traffic to the nucleus to modulate the activity of VP16 in the transcription of the immediate-early genes. In contrast, newly synthesized UL46 is not cleaved and remains within the cytoplasm in association with membranes and is incorporated into the virus during its envelopment at the TGN. If our hypothesis proves to be correct, this will represent one of the first examples as to how the dynamic processing of a specific tegument protein during the assembly process contributes to its functional role within the virus-infected cell. The proposed studies will include two specific aims. The first aim will focus on the characterization of the cleaved UL46 (t-UL46). Our first objective is to identify the cleavage site using both biochemical and genetic approaches. We will then prepare recombinant viruses and expression plasmids that contain genes encoding t-UL46 and well as a gene in which the cleavage site of UL46 has been removed (?t-UL46). Our goal is to use these viruses and expression plasmids to determine the effect of these mutations on the trafficking of t-UL46 and ?tUL-46 as well as their ability to be packaged within virions. Other studies will include defining when the cleavage of UL46 actually occurs during the assembly process. The second aim will focus on determining the functional significance associated with the cleavage of UL46. We will determine if the cleaved t-UL46, which is released from the incoming virus, traffics to the nucleus to enhance the transcriptional activity of immediate-early promoters. The studies proposed within this application may provide a new model for defining molecular mechanisms associated with dynamic events that may occur within the tegument of other human and animal herpesviruses. PUBLIC HEALTH RELEVANCE: The tegument region of the herpesviruses is located between the capsid and envelope and contains approximately 20 proteins;however, there is new evidence that within the virion the tegument is not a static structure. The experiments proposed within this application using herpes simplex virus type 1, represent one of the first studies to define the dynamic interactions that occur among tegument proteins during the maturation of the virus particle. An understanding of these dynamic events that occur within the virus may provide insight as to new targets for disrupting the assembly of this human virus.